(1) Field.
This invention relates to the art of quantitative measurement of protein or polypeptide in solution.
(2) Prior Art.
A variety of methods are available for quantitating proteins present in solutions.
The Lowry procedure is the most commonly employed assay of protein (1) and measures protein with a phenol reagent after alkaline copper treatment. The assay cannot be used on samples containing less than 5 .mu.g/ml of protein; compounds such as K.sup.+, Mg.sup.+2, ethylene diamine tetraacetic acid (EDTA), tris or thiol reagents interfere with the assay.
The biuret reaction using sodium hydroxide and copper sulfate (2) is prone to interference by tris,NH.sub.4.sup.+ and glycerol and is almost two orders of magnitude less sensitive than the Lowry procedure.
Modifications of the above assays have been proposed (3,4) to obviate some of their problems, but the modifications make the assays more time-consuming and introduce additional manipulations.
Ultrasensitive fluorometric assays using fluorescamine have been reported (5,6) but the proteins must be separated from the large number of low molecular weight substances that interfere with the assays. The Kjeldahl assay (7) measures protein nitrogen but is relatively insensitive and very time consuming.
Methods have been reported for quantitating proteins on electrophoretic strips (8) and polyacrylamide gels (9) by staining the protein with Coomassie Brilliant Blue R250 dye. Reisner et al (10) found that Coomassie Brilliant Blue G250 in dilute perchloric acid exhibited a color change when the dye was bound to protein. Bradford (11) has reported a method for quantitating protein in solution based on the color change of Coomassie Brilliant Blue G250 dye in a reagent consisting of 0.01% G250 dye, 4.7% (w/v) ethanol and 8.5% (w/v) phosphoric acid. He found that the ethanol was essential to remove turbidity by solubility the dye/protein complex. U.S. Pat. No. 4,023,933 to Bradford and Williams, is directed to the same process described in the Bradford article (11) and discloses the use of weak acids having a pKa of 0 to 4, preferably 1 to 2, for use with G250 as an assay reagent with an alcohol added to prevent turbidity. The patent specifically states that "Highly ionized acids such as perchloric acid, hydrochloric acid and sulfuric acid cannot be used in the reagent", c. 2, l. 66. A more sensitive dye-binding assay using Amido Schwarz 10B has been reported by Schaffner and Weissman (12) but it requires acid precipitation and collection of the protein on a membrane filter.
The literature citations for the preceding discussion are:
(1) Lowry, O. H., Rosebrough, N.J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265. PA0 (2) Mokrasch, L. C. and McGilvery, R. W. (1956) J. Biol. Chem. 221, 909. PA0 (3) Bensadoun, A. and Weinstein, D. (1976) Anal. Biochem. 70, 241. PA0 (4) Shuster, L. and Schrier, B. K. (1967) Anal. Biochem. 19, 280. PA0 (5) Bohen, P., Stein, S. Imai, K. and Udenfriend, S. (1974) Anal. Biochem. 58, 559. PA0 (6) Nakamura, H. and Pisano, J. J. (1976) Archives Biochem. Biophys. 172, 102. PA0 (7) Wagner, B. C. (1940) Ind. Eng. Chem. 12, 771. PA0 (8) Fazekas de St. Groth, S., Webster, R. G., and Datyner, A. (1963) Biochem. Biophys. Acta. 71, 377. PA0 (9) Diezel, W., Kopperschlager, G. and Hofmann, E., (1972) Anal. Biochem. 48, 617. PA0 (10) Reisner, A. H., Nemes, P. and Bucholtz, C. (1975) Anal. Biochem. 64, 509. PA0 (11) Bradford, M. M. (1976) Anal. Biochem. 72, 248. PA0 (12) Schaffner, W. and Weissmann, C. (1973) Anal. Biochem. 56, 502.